The importance of Th1 and Th2 cells in the normal physiology and pathology of the immune system is well established. Th2-derived cytokines play a central role in the pathophysiology of allergy and asthma. Past studies have documented differences in T cell receptor (TCR)-initiated early signaling between Th1 and Th2 cells. To date, however, no Th2-specific signaling pathways have been identified. The unerlying working hypothesis is that unique TCR-proximal signaling pathways exist in Th2 cells, and that such molecular events represent potential drug targets for selectively interfering with Th2 function. This project will focus on a detailed comparative analysis of early signaling events in Th2 vs. Th1 cells with emphasis on antigen-dependent activation systems. We will: 1) Assess early signaling events during Th2 cell activation and the effects of pharmacological inhibitors/stimulators on the differentiation of naive CD4+ T cells using established clones and T cells from TCR-transgenic mice. These studies will be combined with the use of pharmacological agents which modulate defined T cell signaling events. Effects of these drugs on naive T cell differentiation into Th1 or Th2 cells will be analyzed. 2) Analyze the role of protein tyrosine kinases (PTKs) of the Src. Syk and Tec families in Th1 vs. Th2 cells, and the effects of transient/stable overexpression of wild type or catalytically inactive kinases on activation of the two subsets will be analyzed. 3) Determine the Molecular basis of Ca2+ signaling in Th2 cells, Ca2+ signals in Th2 cells have been reported to differ quantitatively and/or qualitatively from those in Th1 cells. Studies will address the potential role of three alternative Ca2+ signaling pathways mediated by: a) Gq-coupled phospholipase Cbeta: b) sphingosine kinase; or c) CD38. The latter two enzymes generate novel Ca2+-mobilizing second messengers, sphingosine-1-phosphate and cADP ribose, respectively. 4) Analyze the roles of the Ras and MAP kinase signaling pathways in Th2 cells. The function of these pathways (which are critical for Th1 activation) in Th2 cells has not been addressed. We will measure activation of Ras and its downstream Ser/Thr kinases and transcriptional activators, examine the effects of dominant-negative Ras, and assess the role of stress-activated protein kinases in Th2 activation. The information generated by this project is critical for our ability to rationally devise pharmacological treatments designed to selectively inhibit Th2 activation and, thus, interfere with allergic responses.